The present invention relates to novel apple fruit-associated and Thi 1.3:actin fusion promoters, and to heterologous nucleic acid constructs, vectors, kits, and transformation methods employing such promoters. The invention further relates to transgenic plant cells and plants transformed with heterologous nucleic acid constructs comprising an apple fruit-associated or Thi 1.3:actin fusion promoter.
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Transcriptional regulatory sequences or promoters that regulate gene expression in plants are essential elements of plant genetic engineering. Several examples of promoters useful for the expression of heterologous genes in plants are now available (Zhu, et al., 1995; Ni, et al., 1995).
Most promoters are from about 500-1500 bases. Promoters for expressing a heterologous gene sequence in plants can be derived from plant DNA, e.g., the cauliflower heat shock protein 80 (hsp80, Brunke and Wilson, 1993; U.S. Pat. No. 5,612,472), or from other sources, for example, plant viruses e.g., the 35S cauliflower mosaic virus promoter, or bacteria which infect plants, e.g., the nopaline synthase (nos) promoter (Rogers, 1991), the octopine synthase (ocs) promoter (Leisner and Gelvin, 1988) and the mannopine synthase (mas) promoter from Agrobacterium.
Expression of heterologous genes or selected sequences of genes in transgenic plants has typically involved the use of constitutive promoters, which drive the expression of a product throughout the plant at all times and in most tissues (e.g., hsp80), the tomato ubiquitin promoter (Picton, et al., 1993), and the raspberry E4 promoter (U.S. Pat. Nos. 5,783,393; and 5,783,394).
A limited number of inducible and/or tissue specific promoters are known. Promoters that provide fruit-specific expression include the E4 and E8 promoter from tomato (Cordes, et al., 1989; Bestwick, et al., 1995; U.S. Pat. No. 5, 859,330). Another fruit-specific promoter is the tomato 2AII gene promoter. It has been demonstrated that nucleic acid sequences placed under the regulatory control of the 5xe2x80x2 non-coding region of the tomato 2AII gene (Van Haaren, 1993) are preferentially transcribed in developing fruit tissue. Fruit specific regulation of the kiwifruit actinidin promoter has been reported to be conserved in transgenic petunia plants (Lin, et al., 1993).
Differential screening has been used to identify abundant transcripts in developing and ripening fruit. In banana, for example, a cDNA clone encoding a putative thaumatin-like protein is identified as among the most abundant transcripts in ripening fruit, and in kiwifruit a metallothionein-like transcript is identified as very abundant in ripening fruit (Clendennen and May, 1997; Ledger and Gardner, 1994). Abundant transcripts have also been identified in the fruit of grape, cherry, and apple (Fils-Lycaon et al., 1996; Lee et al., 1993).
A transcript was previously identified in Golden Delicious apple (GenBank L15194; Lee, Gardner, and Lay-Yee, 1993, Plant Physiol. 103: 1017) that is abundant in fruit and shows sequence similarity to an auxin-repressed protein (ARP) of unknown function from strawberry.
Apple is a fruit which has been the subject of a great deal of study over the past several decades (Knee, 1993). Ethylene reduction is desired by packers and shippers in order to maintain apples from over-ripening and rotting. Refrigeration, high concentrations of CO2 and low concentrations of O2 are currently being employed to reduce the harmful effects of ethylene during storage. Such methods suffer from the disadvantages that fruits picked at preclimacteric stages (prior to full ripening) respond better to controlled atmosphere conditions than those at mature stages and many varieties of apple suffer from chilling injury and physiological disorders due to controlled atmosphere conditions, rendering them unmarketable.
Ethylene is a plant hormone influencing many aspects of plant growth and development, and is known to play a major role in the ripening process in fruits and vegetables. A large amount of ethylene is also produced following trauma caused by chemicals, temperature extremes, water stress, ultraviolet light, insect damage, disease, or mechanical wounding. In some tissues, exposure to only a small amount of ethylene may cause an avalanche of ethylene production in adjacent plants or plant tissues such as fresh produce. This autocatalytic effect can be very pronounced and lead to loss of fruit quality during transportation and storage.
In plants, the ethylene biosynthetic pathway is an offshoot of the methionine recycling pathway wherein S-adenosylmethionine (SAM) is converted to aminocyclopropane-1-carboxylic acid (ACC) by the enzyme ACC synthase. A bacterial enzyme, S-adenosyl methionine hydrolase (SAMase), not normally present in plant tissue, hydrolyzes SAM, thereby slowing the production of the metabolic precursor of ethylene, ACC.
Stable integration and expression of SAMase in the cells of soft fruits and vegetables has resulted in reduced ethylene production. (See, e.g., Good et al., 1994; Mathews et al., 1995a; Mathews et al., 1995b.) Aminocyclopropane-l-carboxylic acid (ACC)-oxidase and polygalacturonase (PG) promoters have also been isolated from apple and their effects on fruit-specific gene expression evaluated (Atkinson et al., 1998).
A need exists for plant promoters that are selectively functional in particular plant tissues or types of plants and which are capable of providing expression of heterologous genes in the cells of such tissues and plants.
Applicants have identified novel fruit-associated apple promoters, designated in the present application as xe2x80x9cThi-1xe2x80x9d and xe2x80x9cMADS2xe2x80x9d, respectively.
Applicants have also constructed a fusion promoter, designated Thi 1.3:actin, which comprises a 1.3 kb Thi-1 promoter sequence component and a melon actin promoter sequence component.
In one embodiment, the invention provides an isolated nucleic acid molecule comprising an apple fruit-associated xe2x80x9cThi-1xe2x80x9d promoter, a functional portion thereof, or a sequence complementary to it which remains stably bound to the isolated nucleic acid sequence under at least moderate, and optionally, under high stringency conditions.
In exemplary aspects of this embodiment 1.3 kb and 975 bp Thi-1 promoters are provided, as presented in FIG. 1 (SEQ ID NO: 1) and FIG. 2 (SEQ ID NO:2), respectively.
In another embodiment, the invention provides an isolated nucleic acid molecule comprising an apple Thi 1.3:actin fusion promoter or a functional portion thereof, or a sequence complementary to it which remains stably bound to the isolated nucleic acid sequence under at least moderate, and optionally, under high stringency conditions.
An exemplary Thi 1.3:actin fusion promoter has the sequence presented in FIG. 3 (SEQ ID NO:3).
In another embodiment, the invention provides an isolated nucleic acid molecule comprising an apple fruit-associated MADS2 promoter or a functional portion thereof, or a sequence complementary to it which remains stably bound to the isolated nucleic acid sequence under at least moderate, and optionally, under high stringency conditions.
An exemplary apple fruit-associated MADS2 promoter has the sequence presented in FIG. 4 (SEQ ID NO:4).
The invention also provides nucleic acid constructs having a DNA coding sequence under the transcriptional control of an apple fruit-associated or Thi 1.3:actin fusion promoter. The DNA coding sequence is typically heterologous to the promoter and operably linked to the promoter to enable expression of the encoded sequence in fruit cells.
In one respect, an apple fruit-associated or a Thi 1.3:actin fusion promoter of the present invention can be used to modulate ethylene production in transformed fruit cells and thereby alter the ripening phenotype of transgenic fruit composed of such fruit cells.
In this embodiment of the invention, the apple fruit-associated promoters and Thi 1.3:actin fusion promoter described herein are employed in a method for prolonging ripening and delaying senescence of fruit from a fruit-bearing plant. In this method, a transgenic plant containing a promoter of the present invention operably linked to a heterologous DNA coding sequence is grown to the fruit-bearing stage, at which point the heterologous DNA coding sequence is expressed in the fruit of the transgenic fruit-bearing plant.
In particular, the heterologous DNA sequence encodes a product capable of reducing ethylene biosynthesis when expressed in plant cells, e.g., S-adenosyl methionine hydrolase (SAMase), amino-cyclopropane-1-carboxylic acid (ACC) deaminase, ACC oxidase antisense molecule, ACC synthase antisense molecule, ACC oxidase cosuppression molecule, and ACC synthase cosuppression molecule. Fruit produced by these transgenic plants have a modified ripening phenotype. A modified ripening phenotype refers to an alteration of the rate of ripening (e.g., prolonged ripening and delayed senescence) of a transgenic fruit relative to corresponding (ie., non-transgenic) wild-type fruit.
In another embodiment, an apple fruit-associated or Thi 1.3:actin fusion promoter of the invention is used to control expression of a DNA coding sequence such as a pathogenesis related gene, e.g polygalacturonase inhibiting protein (PGIP), glucanase and chitinase.
Additional exemplary DNA coding sequences include, but are not limited to sequences which encode, thaumatin, sucrose phosphate synthase, invertase, lycopene cyclase, antimicrobial peptides, invertase, antisense polyphenol oxidase, antisense polyphenol peroxidase, and antisense pectate lyase.
The invention further includes a method for producing a transgenic plant such as a fruit-bearing plant. In this method, a chimeric gene, typically carried in an expression vector allowing for selection in plant cells, is introduced into progenitor cells of selected plant. These progenitor cells are then grown to produce a transgenic plant bearing fruit.
The methods and results described herein demonstrate tissue-associated regulation of gene expression in transgenic plants. The tissue-associated promoters of the present invention include a DNA sequence that regulates transcription of a heterologous nucleic acid coding sequence to which it is operably linked.
The present invention also includes the use of any of the above promoters in plant transformation vectors. Such vectors can be used in any plant cell transformation method, including Agrobacterium-based methods, electroporation, microinjection, and microprojectile bombardment. These vectors may form part of a plant transformation kit. Other components of the kit may include, but are not limited to, reagents useful for plant cell transformation.
In another embodiment, the invention includes a plant cell, plant tissue, transgenic plant, fruit cell, whole fruit, seeds or calli containing any of the above-described promoters, chimeric genes and the corresponding expressed gene products.
These and other objects and features of the invention will become more fully apparent when the following detailed description is read in conjunction with the accompanying figures and examples.